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eCAM 2004;1(1)8391

Original Article

Combination Effects of Herbs in a Multi-herbal Formula: Expression


of Juzen-taiho-tos Immuno-modulatory Activity on the Intestinal
Immune System
Hiroaki Kiyohara1,2, Tsukasa Matsumoto1,2 and Haruki Yamada1,2
1Kitasato

Institute for Life Sciences, Kitasato University, Tokyo and 2Oriental Medicine Research Center, The Kitasato
Institute, Tokyo, Japan
Herbal formulas of traditional Japanese (Kampo), Chinese and Korean medicines usually comprise
multiple herbs in a single formula. These medicines are expected to show their clinical effects by
chemical, pharmacological and pharmaceutical combination effects of multi-herbs. However, little
effort has been made so far to scientifically clarify the nature of such combination effects. Interestingly, for example, though a Kampo medicine Juzen-taiho-to (Shi-Quan-Da-Bu-Tang in Chinese)
stimulates the immune functions of Peyers patch cells, none of its single component herbs shows such
activity. We thus examined the combination effect of herbs in the Juzen-taiho-to formula for the
expression of its immuno-stimulating activity. Juzen-taiho-to, a composite formula of 10 herbs, has
been generally considered to comprise two kinds of basic formula, each of which consists of four different herbs in addition to two others. The combinations of herbs based on these two basic formulas
were evaluated for their stimulating activities on cytokine production from murine Peyers patch cells
both in vitro and ex vivo. Combined decoction of six among 10 herbs in Juzen-taiho-to is crucial for
the expression of its stimulating activity on Peyers patch cells. 3D-HPLC analysis of the ingredients in
the fractions from the combined decoctions indicated that, in addition to quantitative changes of
ingredients, alterations occur in their chemical composition by decoction of different herbs. The stimulating activity of Juzen-taiho-to on Peyers patch cells results from the combination effect of its six
essential component herbs. This combination effect is based on physicochemical interactions among
the ingredients of the component herbs.
Keywords: combination effect lignincarbohydrate complexes Peyers patch cells

Introduction
A number of herbal formulas of traditional Japanese medicines (Kampo), and Chinese and Korean ones as well, are
characterized by the use of mixtures of several herbs (multiherbs) in a single formula. These multi-herb formulas are
usually prepared in various dosage forms such as decocted
extracts, pills, powders, tablets etc. in a traditional way.
Therefore, particularly in the case of decocted extracts with
boiling water, one has to expect the possibility that some
chemical interactions take place among natural constituents
existing in the component herbs of the formula during decoction. Decoction may change the extraction rates of the active
ingredients and/or produce new artificial substances, which

For reprints and all correspondence: Haruki Yamada, Kitasato Institute for
Life Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
E-mail: yamada@lisci.kitasato-u.ac.jp

may then exhibit new pharmacological activities. These types


of pharmaceutical actions, derived from the combination of
multiple herbs in a formula, are called chemical combination
effects. Meanwhile, it has also been postulated that in multiherbal formulas, the pharmacological activities of one single
herb is either potentiated or prolonged, and/or its adverse
effects reduced, due to synergistic or antagonistic effects, by
addition of other herbs (1). These complex phenomena are
supposed to emerge as a number of different active ingredients interact with each other, acting on different target
systems in the body. It is also possible that certain pharmacokinetic situations such as concentrations in blood and
metabolism of ingredients are altered in the presence of
other ingredients which may affect drug transporters and
drug metabolizing enzymes. These types of pharmacological
action are called either pharmacological combination
effects or pharmaceutical combination effects. However,
because of the sheer complexity of the phenomena, not many
Oxford University Press 2004

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Combination effects of multi-herbs in Juzen-taiho-to

Table 1. Component herbs of Juzen-taiho-to and its basic formulations


Component herb

Juzen-taiho-to

SMT

SKT

alone

with ASR

with CC

alone

with ASR

with CC

SMT and SKT


with ASR and CC

Rehmanniae Radix (3 g)

Paeoniae Radix (3 g)

Cnidii Rhizoma (3 g)

Angelicae Radix (3 g)

Atractylodis lanceae Rhizoma (3 g)

Ginseng Radix (3 g)

Poria (3 g)

Glycyrrhizae Radix (3 g)

Astragali Radix (ASR, 1.5 g)

Cinnamomi Cortex (CC, 3 g)

Intestinal immune system


modulating activity (in vitro)

*
*

*
*

Asterisks indicate presence of herb in the formulation listed at the top of each column.
Full activity.
No activity.
Statistically significant activity.

researchers have tackled this problem by performing actual


experiments to scientifically elucidate these interesting
effects.
Juzen-taiho-to (Shi-Quan-Da-Bu-Tang in the Chinese pronunciation) is one famous formula in Kampo, Chinese and
Korean medicines, which comprises 10 different kinds of single herbs. Juzen-taiho-to has been clinically used in Japan for
the treatment of patients recovering from surgery or suffering
from chronic disease, to promote improvement of their debilitated general condition. By pharmacological evaluation
using a pharmaceutically homogeneous preparation (TJ-48)
of Juzen-taiho-to, it has been found that the herbal formula
not only affects the systemic immune functions of T and B
cells, macrophages and NK cells, but also those of the hematopoietic and intestinal immune systems (210). It has been
reported that several kinds of macromolecular polysaccharides, especially lignincarbohydrate complexes, and numerous kinds of low molecular weight components are its active
ingredients (915). Some of these active ingredients have also
been identified in some of its single herb components (11,12).
Although the lignincarbohydrate complexes obtained from
TJ-48 could show a potent modulatory activity on the intestinal immune system, lignincarbohydrate complexes from any
of the 10 single herbs in TJ-48 failed to show such activity,
indicating the existence of certain combination effects among
multiple herbs in the formula.
We therefore aimed in the present study to evaluate the
potential combination effects of herbs in the Juzen-taiho-to
formula in its modulatory activity on the intestinal immune
system.

Subjects and Methods


Materials
Spray-dried extract preparation (TJ-48) of Juzen-taiho-to
was kindly supplied by Tsumura & Co. (Tokyo, Japan). TJ-48
was prepared as described previously (14). Astragali Radix
(roots of Astragalus membranaceus Bunge), Rehmanniae
Radix (roots of Rehmania glutinosa Libosch var. purpurea
Makino), Paeoniae Radix (rhizomes of Paeonia lactiflora
Pall), Cnidii Rhizoma (rhizomes of Cnidium officinale
Makino), Atractylodis Lanceae Rhizoma (rhizomes of
Atractylodes lancea DC), Ginseng Radix (roots of Panax
ginseng C.A.Meyer), Poria (fungi of Poria cocos Wolf) and
Glycyrrhizae Radix (roots of Glycyrrhiza uralensis Fisch et
DC) were purchased from Uchida-Wakan-Yaku Co. (Tokyo,
Japan) and Cinnamomi Cortex (barks of Cinnamomum cassia Blume) and Angelicae Radix (roots of Angelica acutiloba
Kitagawa) were obtained from Tochimoto-Tenkaido (Osaka,
Japan).
Preparation of LigninCarbohydrate Complexes
Containing Fraction (F-3) From TJ-48 and the
Corresponding Fractions (F-3-related Fractions) From
Each Single Herb
The lignincarbohydrate-complex-containing fraction, which
we call F-3, was prepared from TJ-48 according to the procedure described previously (14). The corresponding fractions (F-3-related fractions) were also obtained from each
single herb and the mixture of single herbs was obtained in a
similar manner. Briefly, 10 g of each component herb was
decocted with 100 ml of water to half volume, and the residual materials were re-decocted by the same procedure. After
the combined extract was concentrated to 50 ml by evaporation, 4 vol of EtOH was added to the solution, and the

eCAM 2004;1(1)

resulting precipitate was removed. The resulting supernatant


was evaporated to dryness, and hydrophobic low-molecularweight ingredients were removed by refluxing with MeOH
(100 ml, 1 h). The MeOH-insoluble materials were dialyzed
by cellulose dialysis tube (molecular weight cut off: 12 000
14 000) against distilled water for 4 days. The dialyzable portion was recovered and evaporated to obtain water-soluble
and dialyzable fraction as F-3-related fractions of the component herbs.
The two kinds of basic formulaShikunshi-to (SKT) (SiJun-Zi-Tang in Chinese pronunciation) and Shimotsu-to
(SMT) (Si-Wu-Tang in Chinese pronunciation)and the
corresponding combinations of single component herbs of
Juzen-taiho-to were prepared as shown in Table 1. These formulas and combinations were decocted with 600 ml of water
to half volume, and the residual materials were re-decocted
by the same procedure. The combination of two single herbs,
Astragali Radix (ASR) (3 g) and Cinnamomi Cortex (CC)
(3 g) was also decocted in the same manner. The resulting
extracts were concentrated to 50 ml, and the water-soluble
and dialyzable fractions from these decoctions were prepared
as F-3-related fractions by the same procedure as described
above, using MeOH extraction, EtOH precipitation and
dialysis.
Measurement of Intestinal Immune System Modulating
Activity
In vitro and in vivo activities were measured as described
previously (10,15). For in vivo experiments, suspensions of
Peyers patch cells in RPMI-1640 medium (Gibco, Grand
Island, NY) supplemented with 5% fetal bovine serum
(RPMI 1640-FBS) were prepared at day 8 from the small
intestine of C3H/HeJ mice (female, 68 weeks old, SLC,
Japan), who had been orally administered with F-3-related
fractions (150 mg/kg/day) or distilled water (control) for one
week. Aliquots (200 l) of the cell suspension (12 106
cells/ml in RPMI 1640-FBS) of Peyers patch cells were
cultured in 96-well flat bottom microtiter plates for 6 days
at 37C in a humidified atmosphere of 5% CO295% air.
For in vitro experiments, 20 l of aqueous solutions of
samples or distilled water were cultured for 6 days with 180 l
of cell suspension of Peyers patch cells, prepared from the
small intestine of C3H/HeJ mice. The resulting culture
supernatants (50 l) obtained by in vitro and in vivo experiments were further cultured with bone marrow cell suspension (2.55.0 105 cells/ml), which were prepared from
C3H/HeJ mice, in a humidified atmosphere of 5% CO2
95% air for 6 days. At 530 h prior to culture termination,
20 l of Alamar Blue solution (Alamar Bio-Sciences Inc.,
Sacramento, CA) was added to each well, and the cells were
then continuously cultured. The fluorescence intensity was
measured by Fluoroscan II (Labosystems) at an excitation
wavelength of 544 nm and an emission wavelength of 590
nm. The modulatory activity on the intestinal immune system
was expressed as fluorescence intensity compared to that of

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control. In vitro activity of each sample was obtained from


four experiments in all case, and in vivo activity from 36
experiments. The results of fluorescence intensity are varied
depending on the conditions of Peyers patch cells and bone
marrow cells in this assay system, and are comparable among
the results in the same culture plate.
Statistics
All results are expressed as the mean S.E. The differences
between the control and the treatments in these experiments
were tested for statistical significance by Students t-test (in
vitro) and Welchs t-test (in vivo). The differences between
the treatments were tested by Scheffe test (in vitro). A value
of P < 0.05 was considered to indicate statistical significance.
Analytical Methods
Total carbohydrate and uronic acid were determined by
phenolH2SO4 and m-hydroxybiphenyl methods, respectively (16,17). Lignin content was analyzed by Bradfords
method with Bio-Rad dye (Bio-Rad) (18), because the color
reagent in this method cross-reacts with standard lignin (H.
Kiyohara, unpublished data). Component sugars of the
samples were analyzed as trimethylsilyl methylglycoside
derivatives by GLC as described previously (15).
F-3-related fraction prepared from SKT and SKT with
ASR and CC were fractionated on Sephadex G-25 equilibrated with H2O and the eluted materials were analyzed for
carbohydrate, uronic acid and lignin-like molecules by the
phenolH2SO4 method, the m-hydroxybiphenyl method and
measurement of UV absorption at 280 nm. Three dimensional (3D-)HPLC analyses were performed on Agilent 1100
Series HPLC system equipped with photodiodarray detector
and TSK gel-ODS 80Ts column (4.6 250 mm, Toso Co.
Ltd). The 3D-HPLC conditions were as follows: flow rate at
0.8 ml/min; 10 mM H3PO4-CH3CN = 95:55:95 (60 min,
linear gradient); monitored from 200 to 600 nm.

Results
The fractions (F-3) containing lignincarbohydrate complexes, which were prepared from Juzen-taiho-to (TJ-48),
and F-3-related fractions from hot water extracts of each of
the 10 single herbs of TJ-48, were compared for their modulatory activity on the intestinal immune system. Although F-3
obtained from TJ-48 exhibited a potent activity, none of the
F-3-related fractions from each single component herb had
such activity (Fig. 1). When F-3-related fractions of the 10
single herbs were mixed in equal amounts, or in ratios according to their yields, and were measured for activity, these
mixed F-3-related fractions also failed to express any activity
(Fig. 2A). These results clearly indicate that the combined
decoction of over two kinds of single herbs in the formula of
Juzen-taiho-to is necessary for obtaining F-3 possessing
activity. To further analyze this interesting phenomenon,
identification of the essential combination is necessary. How-

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Combination effects of multi-herbs in Juzen-taiho-to

Figure 1. Intestinal immune system modulating activity of lignincarbohydrate complexes containing fractions (F-3) prepared from Juzen-taiho-to (TJ-48) and
F-3-related fractions from component herbs. Filled squares, 10 g/ml; open squares, 100 g/ml.

Figure 2. (A) Intestinal immune system modulating activity of combined F-3-related fractions, which were prepared by mixing of F-3-related fractions
obtained from each of the 10 component herbs in equal ratios (a) and in ratios according to their yields (b). (B) Intestinal immune system modulating activity
of various F-3-related fractions prepared from decoctions of SMT or SKT with or without ASR or CC, and from the decoction of the combination of SMT and
SKT. Filled squares, 10 g/ml; open squares, 100 g/ml.

ever, in identifying the essential combination of single herbs


for the expression of activity, it would be extremely inefficient
if F-3-related fractions were prepared from random combinations of the 10 single herbs: numerous kinds of over 100
combinations have to be evaluated.

The formula of Juzen-taiho-to is generally recognized in


the theory of Oriental herbal medicine to comprise two kinds
of basic formulas, SMT (consisting of four single herbs:
Rehmanniae Radix, Paeoniae Radix, Cnidii Rhizoma and
Angelicae Radix) and SKT (consisting of four single herbs:

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Figure 3. (A) Intestinal immune system modulating activity of various F-3-related fractions prepared from decoctions of SKT with ASR alone or with ASR and
CC combined, and from decoction of the combination of ASR and CC. (B) Effect of omitting each component herb from the combination of SKT with ASR
on intestinal immune system modulating activity of their F-3-related fractions. Filled squares, 10 g/ml; open squares, 100 g/ml. *P < 0.05 versus control.
N.S., not significant.

Atractylodis Lanceae Rhizoma, Ginseng Radix, Poria and


Glycyrrhizae Radix) in addition to ASR and CC (Table 1).
Therefore, we selected multi-herbs according to the formulas
of SMT, SKT or SMT combined with SKT, each of which
were decocted, and F-3-related fractions were prepared to
measure activity. We found that none of the F-3-related fractions showed activity, however, which indicated that the
multi-herbs within eight kinds of single herb based on the formulas of SMT and SKT do not contribute to the expression
of activity (Fig. 2B, Table 1). When other single herbs, ASR
or CC, were also combined with the SMT formula, F-3related fractions prepared from the combined decoctions of
herbs in these combinations did not show any intestinal
immune system modulating activities either (Fig. 2B, Table
1). However, although F-3-related fraction obtained from the
combined decoction of herbs in SKT with CC showed no
activity, the F-3-related fraction prepared from SKT with
ASR did exhibit a statistically significant activity (Fig. 2B,
Table 1). It was also discovered that the combined decoction
of CC with the five single herbs in SKT with ASR significantly increased the activity of its F-3-related fraction to the
same level as F-3 from TJ-48 (Fig. 3A, Table 1). In contrast,
the F-3-related fraction prepared from the combined decoction of two kinds of single herbs, ASR and CC, had no such
activity (Fig. 3A).
We then orally administered F-3-related fractions (150
mg/kg/day), prepared from the combined decoction of SKT
and from that of SKT with ASR and CC, to C3H/HeJ mice
for 1 week. It was discovered that the F-3-related fraction

from the combination of SKT with ASR and CC expressed a


statistically significant activity to modulate the intestinal
immune system ex vivo (Fig. 4). However, the F-3-related
fraction from the formula of SKT alone did not show the
activity as observed in the in vitro experiment. When each
one single herb, excluding ASR, was removed from the set of
five single herbs in the formula of SKT with ASR, and F-3related fractions were prepared from the decoctions of these
modified combinations, none of the resulting F-3-related
fractions showed the activity (Fig. 3B). This result strongly
indicates that the combined decoction of five single herbs
(Atractylodis Lanceae Rhizoma, Ginseng Radix, Poria, Glycyrrhizae Radix and ASR) is required for the expression of
the modulatory activity on the intestinal immune system.

Figure 4. Ex vivo intestinal immune system modulating activity of F-3related fraction prepared from SKT or SKT with ASR and CC.

88

Combination effects of multi-herbs in Juzen-taiho-to

Figure 5. Gel filtration patterns on Sephadex G-25 of F-3-related fractions prepared from the formulation of SKT (A) or the combination of SKT with ASR
and CC (B). Closed circles, carbohydrate (490 nm); open circles, uronic acid (520 nm); closed triangles, phenolics or protein (280 nm).

We subsequently performed the following experiment to


comparatively analyze the difference of active components in
the F-3-related fractions among different combinations of
herbs. F-3-related fractions were first prepared by combined
decoction of either the herbs of the SKT formula or SKT
with ASR and CC, then fractionated by Sephadex G-25 gel
filtration. Both of these F-3-related fractions gave small
amounts of the highest-molecular-weight fraction (F-3-1),
the intermediate size fraction (F-3-2), and large amounts of
lower- and lowest-molecular-weight fractions (F-3-3 and F-34) (Fig. 5). All the subfractions from the F-3-related fraction
of SKT with ASR and CC showed significantly higher intestinal immune system modulating activity than the corresponding subfractions from the F-3-related fraction of SKT (Fig.
6A). Among the subfractions from SKT with ASR and CC,
F-3-1 showed the most potent activity in a dose-dependent
manner (Fig. 6A and B), and F-3-4 had the second most
potent activity, whereas F-3-2 and F-3-3 showed the weakest
activities (Fig. 6A). When yields of these subfractions were
compared, those of F-3-1 from SKT, and SKT with ASR and
CC were similar, whereas the yield of F-3-4 from SKT with
ASR and CC was almost twice as high as that of F-3-4 from
SKT (Table 2). We considered that this result can explain one
of the reasons why only the F-3-related fraction from SKT
with ASR and CC showed the activity. It is also suggested
that the combined decoction of the six single herbs from SKT
with ASR and CC may increase the extraction efficiency of
the active ingredients in F-3-4. It has been reported that the
F-3-1-related fraction from TJ-48 comprises lignincarbohydrate complexes as the active ingredients for the modulatory

activity on the intestinal immune system (14). Carbohydrate


contents of F-3-1 from SKT, and SKT with ASR and CC
were similar, and component sugar analysis also indicated
that both F-3-1 consisted mainly of glucose (Table 2). However, the lignin content of F-3-1 from SKT was remarkably
higher than that from SKT with ASR and CC (Table 2). The
presence of active and inactive lignin molecules has been
suggested for modulation of the intestinal immune system
(14), and the compositions of lignin-containing ingredients is
assumed to be different between the F-3-1 subfraction from
SKT and that from SKT with ASR and CC.
Components of F-3-4 from both multi-herbs seemed to be
low molecular weight substances. In order to evaluate the difference of constituents between the subfractions, F-3-4 from
SKT and SKT with ASR and CC, F-3-4 from both multiherbs were compared by 3D-HPLC. Because most of the
ingredients in F-3-4 had maximal absorption near 250 and
280 nm, elution profiles were analyzed by single wavelength
at 250 or 280 nm (Fig. 7). Elution profiles of F-3-4 from SKT
and that from SKT with ASR and CC were similar, with little
differences in peak areas of constituents observed at 250 nm.
However, the areas of the peaks having retention times near
26.0 and 32.2 min were significantly higher in F-3-4 from
SKT with ASR and CC than in F-3-4 from SKT in analyses at
280 nm, and the peak having the retention time near 28.0
min in F-3-4 from SKT disappeared in that from SKT with
ASR and CC.

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89

Figure 6. (A) Intestinal immune system modulating activity of subfractions of F-3-related fractions prepared from the formulation of SKT alone or from the
combination of SKT with ASR and CC. Filled squares, 10 g/ml; open squares, 100 g/ml. *P < 0.05 versus control. (B) Dose-dependent intestinal immune
system modulating activity of F-3-1 from Fig. 5. Open circles, F-3-1 from SKT; filled squares, F-3-1 from SKT with ASR and CC. *P < 0.005 versus data of F3-1 from SKT.

Table 2. Property of subfractions obtained from F-3 of the prescriptions of SKT, SKT with ASR and CC and TJ-48
SKT

SKT with ASR and CC

TJ-48
F-3-1-1A

F-3-1

F-3-2

F-3-3

F-3-4

F-3-1

F-3-2

F-3-3

F-3-4

Yield (%)

9.2

9.2

61.1

20.4

7.6

4.2

38.8

49.4

Carbohydrate content (%)*

79.2

84.9

n.d.

Relative lignin content (%)

127.8

57.0

54.9

Arabinose

8.6

9.0

16.5

Rhamnose

3.5

2.9

7.0

Fucose

0.02

0.06

1.8

Xylose

1.8

2.1

4.2

Glucuronic acid

9.2

3.6

3.4

Galacturonic acid

7.7

4.8

11.5

Mannose

4.0

8.0

5.8

Component sugars (mol. %)

Galactose

8.0

6.0

15.6

Glucose

57.3

63.2

32.4

*Glucose was used as standard.


available lignin was used as standard.
Reported in Kiyohara et al. (14).
n.d., not determined.
Commercially

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Combination effects of multi-herbs in Juzen-taiho-to

Figure 7. HPLC patterns of F-3-4 prepared from SKT with ASR and CC (A, C) and from SKT alone (B, D). (A, B) Monitored at 250 nm; (C, D) Monitored
at 280 nm. The peaks with the arrows were significantly different between both F-3-4.

Discussion
In pharmacological study of the formulas in Kampo medicine, previous investigators have noted interesting phenomena when several single herbs are combined to be decocted
into one formula. For example, Hosoya has indicated that
antitussive action of the hot water extract of Ephedra Herba
is enhanced by the combined decoction of this herb with
three other kinds of single herbs, such as Armeniacae Semen,
Glycyrrhizae Radix and Gypsum Fibrosum, while each of its
hot water extracts alone has no antitussive action (1). It was
also found that the duration and strength of antitussive effect
of Ephedra Herba were prolonged and potentiated by this
combined decoction compared with Ephedra Herba alone.
As another example, a dried extract from one formula of
Kampo medicine, Sho-saiko-to-go-Keishi-ka-shakuyaku-to
(TJ-960), which consists of 10 kinds of single herbs, shows
anti-convulsant action on seizures induced by the administration of pentetrazole to guinea pigs. However, if any single
herb except Zingiberis Rhizoma from the multi-herbs of this
formula is omitted, the anti-convulsant activity of the hot
water extract from the complete formula disappears, suggesting that the combined decoction of nine single herbs in the
formula of TJ-960 is necessary for expression of the activity
(1). However, what constitutes the chemical, pharmacological and pharmaceutical basis for such phenomena of combination effects remains obscure.

The present results revealed that not all of the 10 single


herbs in the formula of Juzen-taiho-to are required for the
expression of its immuno-modulatory activity on the intestinal immune system. The combined decoction of six single
herbs according to the formula of SKT (Atractylodis Lanceae
Rhizoma, Ginseng Radix, Poria and Glycyrrhizae Radix)
with ASR and CC is crucial for the activity of the F-3-related
fraction to reach the same level as the F-3 of TJ-48. It is also
suggested that the combined decoction of five single herbs
(Atractylodis Lanceae Rhizoma, Ginseng Radix, Poria,
Glycyrrhizae Radix, ASR) is essentially required for the
expression of statistically significant activity. Although the
F-3-related fraction prepared from the combined decoction
of herbs due to SKT with ASR and CC also showed significant activity to modulate the intestinal immune system even
in vivo, it was not as remarkable as that observed in the in
vitro experiments. The experimental conditions such as dose
of F-3-related fraction and period of oral administration
need to be optimized in further study in order to evaluate
the combination effect of the six kinds of single herbs due
to SKT with ASR and CC in vivo.
The present study suggests that the SMT formula does not
contribute to the expression of the activity, because its F-3related fraction after combined decoction did not show the
activity at all, even if ASR or CC was further added to the
multi-herbs of SMT.

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Juzen-taiho-to is known to have multifunctional properties


in clinical applications. For example, Saiki et al. found that
the combined decoction of the component herbs of SMT
alone contribute to the expression of anti-metastatic activity
of Juzen-taiho-to, but not that of the component herbs of
SKT (19). Therefore, we suggest that SMT may play a different role from SKT for the expression of clinical effects of
Juzen-taiho-to. The present study evaluated the effects of the
combination of single herbs of Juzen-taiho-to only from the
viewpoint of its modulating activity on the Peyers patch cells
in the intestinal immune system. We would therefore not
exclude the possibility that the component herbs of SMT
may play a role in modulation of the functions of other
immune cells in the intestinal immune system.
The combined F-3-related fraction prepared by mixing of
fractions containing lignincarbohydrate complexes from 10
respective single herbs of Juzen-taiho-to failed to express the
activity. This observation strongly suggests that the combined
decoction of all essential herbs is quite important to bring
about the combination of ingredients required for the expression of the activity. The present study indicates that yields of
some low molecular weight ingredients were changed in
decoction by the addition of ASR and CC to the multi-herbs
of SKT. Therefore, we propose that the extraction efficiency
of some active ingredients may be enhanced by the addition
of ASR or ASR and CC to SKT.
The 3D-HPLC analysis clearly showed that some ingredients are either increased or disappear on the addition of ASR
and CC to SKT; however, it is still not known whether the
ingredients are decreased by the reaction with other ingredients in the herbs of SKT with ASR and CC. The highest
molecular weight fraction, F-3-1, was assumed to contain
lignincarbohydrate complexes from the results of the study
on the water-soluble and dialyzable fraction (F-3) of Juzentaiho-to for their modulatory activities on the intestinal
immune system (14). The present results also allow us to postulate that even the relatively high molecular weight ingredients might also be changed in their content and/or structure
by the combined decoction of multi-herbs. However, details
of this change will require future clarification. The clarification must await further study on both high and low molecular
weight ingredients regarding the precise events taking place
on them during decoction of multi-herbs.

Acknowledgments
Part of the present work was supported by a fund from
Tsumura & Co., Tokyo. Part of the present work was also
supported by The 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan. We thank Ms F. Honma and Ms E.
Yoshida for their technical assistance.

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References
1. Hosoya E. Scientific reevaluation of Kampo prescriptions using modern
technology. In Hosoya E, Yamamura Y. (eds) Resent Advances in the
Pharmacology of Kampo (Japanese herbal) Medicines. Tokyo, Japan:
Excerpta Medica 1988;1729.
2. Komatsu Y, Takemoto N, Maruyama H, Tsuchiya H, Aburada M,
Hosoya E, Shinohara S, Hamada H. Effects of Juzen-taiho-to on the
anti-SRBC response in mice. Jpn J Inflamm 1986;6:4058.
3. Maruyama H, Kawamura H, Takemoto N, Komatsu Y, Aburada M,
Hosoya E. Effect of Juzen-taiho-to on phagocytes. Jpn J Inflamm 1988;
8:4615.
4. Takemoto N, Maruyama H, Kawamura H, Komatsu Y, Aburada M,
Hosoya E. Mitogenic activity of Juzen-taiho-to (TJ-48) on murine
lymphoid cells. Jpn J Inflamm 1989;9:13740.
5. Takemoto N, Kiyohara H, Maruyama H, Komatsu Y, Yamada H,
Kawamura H. A novel type of B-cell mitogen isolated from JuzenTaiho-To (TJ-48), a Japanese traditional medicine. Int J Immunopharmacol 1994;16:91929.
6. Matsumoto T, Sakurai MH, Kiyohara H, Yamada H. Orally administered decoction of kampo (Japanese herbal) medicine, Juzen-Taiho-To
modulate cytokine secretion and induces NKT cells in mouse liver.
Immunopharmacology 2000;46:14961.
7. Kawamura H, Maruyama H, Takemoto N, Komatsu Y, Aburada M,
Ikehara S, Hosoya E. Accelerating effect of Japanese kampo medicine
on recovery of murine hemopoietic stem cells after administration of
mitomycin C. Int J Immunother 1989;5:3542.
8. Sugiyama K, Yokota M, Ueda H, Ichio Y. Protective effects of kampo
medicines against cis-diaminedichloroplatinum (II)-induced nephrotoxicity and bone marrow toxicity in mice. J Med Pharm Soc Wakan-Yaku
1993;10:7685.
9. Hisha H, Yamada H, Sakurai MH, Kiyohara H, Li Y, Yu CZ, et al.
Isolation and identification of hematopoietic stem cell-stimulating
substances from Kampo (Japanese herbal) medicine, Juzen-Taiho-To.
Blood 1997;90:102230.
10. Hong T, Matsumoto T, Kiyohara H, Yamada H. Enhanced production
of hematopoietic growth factors through T cell activation in Peyers
patches by oral administration of kampo (Japanese herbal) medicine,
Juzen-Taiho-To. Phytomedicine 1998;5:35360.
11. Kiyohara H. Elucidation of pharmacologically active polysaccharides
from Kampo medicines and component herbs. In Ageta H, Aimi N,
Ebizuka Y, Fujita T, Honda G. (eds) Towards Natural Medicine
Research in the 21st Century. Tokyo, Japan: Elsevier 1998;16171.
12. Yamada H. The role of bioactive polysaccharides in Kampo medicines.
In Watanabe H, Shibuya T. (eds) Pharmacological Research on Traditional Herbal Medicine. Amsterdam, the Netherlands: Harwood
Academic Publishers 1999;17996.
13. Yamada H, Kiyohara H. Complement-activating polysaccharides from
medicinal herbs, In: Wagner H. (ed.) Immunomodulatory Agents from
Plants. Basel, Switzerland: Birkhauser Verlag 1999;161202.
14. Kiyohara H, Matsumoto T, Yamada H. Lignincarbohydrate complexes: Intestinal immune system modulating ingredients in kampo
(Japanese herbal) medicine, Juzen-Taiho-To. Planta Med 2000;66:20
4.
15. Kiyohara H, Matsumoto T. Yamada H. Intestinal immune system
modulating polysaccharides in a Japanese herbal (Kampo) medicine,
Juzen-Taiho-To. Phytomedicine 2002;9:61424.
16. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric
method for determination of sugars and related substances. Anal Chem
1956;28:3506.
17. Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acid. Anal Biochem 1973;54:4849.
18. Bradford MM. A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:24854.
19. Onishi Y, Yamaura T, Tauchi K, Sakamoto T, Tsukada K, Nunome S,
Komatsu Y, Saiki I. Expression of the anti-metastatic effect induced by
Juzen-taiho-to is based on the content of Shimotsu-to constituents.
Biol Pharm Bull 1998;21:7615.
Received October 23, 2003; accepted January 19, 2004

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